Abstract: The strains producing acid were isolated in this study. The transparent ring method on MRS-Ca CO₃ medium plates were used to isolate a bacterial strain producing more acid from the 10 ~30 d breast milk after giving birth. The optimal strain was identified by morphological observation, physiological and biochemical tests and 16 S r DNA sequences, and it presented Gram positive, producing more lactic acid, rod-shaped and non-spore forming. The results of physiological and biochemical tests were consistent with Lactobacillus rhamnosus, named Z5. When the strain was incubated in artificial gastric juice and intestinal juice with p H3.0 for 6 h, the survival rates were more than 90%. After 6 h incubation in 0.15 g/L bile salt, the survival rate for the strain was 76.14%.The growth of Salmonella pullorum and Escherichia coli were inhibited by the cultures of the strain Z5 and the antibacterial circle diameter was greater than 20 mm.The strain Z5 could effectively adhered to the Caco-2cells and the average number of adhesion was 7.54 × 105cfu/m L. These results suggested that Lactobacillus rhamnosus Z5 isolating from human breast milk showed the high survival rate in gastrointestinal environments and could be a good candidate for probiotic application.